Project Summary: The development of aptamers is a topic of wide interest due to their potential use in therapeutic applications, as biosensors, or as biochemical tools, among others. This proposal focuses on the use of 8- oxo-7,8-dihydroguanosine (8-oxoG) as a novel building block in the discovery of aptamers of RNA with new properties such as enhanced affinity, distinct specificity (from that of canonical analogues), and/or new structural features. The approach relies on 1) developing methodology that permits a selection process for libraries of RNA containing 8-oxoG; 2) the use of known scaffolds as initial models with novel specificity and structural motifs, where the model uses the aptamer for theophylline; and 3) the development of structural probing techniques to assess the impact of 8-oxoG on structure, locally and globally, and function of RNA. The incorporation of 8-oxoG within aptamers of RNA will affect their structure and function in a way that expands the ?toolkit? of available nucleobases in the design of novel therapeutic agents. 8-oxoG is a well-known modification that occurs under oxidative stress in DNA and RNA. Its inherited features include the formation of distinct H-bonding networks as well as conformational changes that, in turn lead to altered base pairing and H-bonding interactions with various proteins. This proposal takes advantage of those properties with the aim to control the structure and function of RNA and gain access to constructs with new properties. Thus, expanding the toolkit available upon designing RNA constructs with distinct recognition capabilities towards small molecules and/or proteins The methodology to develop a selection process relies on establishing the unique reactivity between RNA containing 8-oxoG and various reverse transcriptases (RTs), followed by signal amplification via PCR and readouts that rely on the synthesis of cDNA with sequences of varying ratios that depend on the RT used. To validate the working hypothesis that the presence of 8-oxoG within RNA affects its functional outcomes, known scaffolds will be modified followed by structural and functional analyses. The aptamer for theophylline will be used as model to rationally assess changes in structure and specificity, where a combination of circular dichroism, microscale thermophoresis, and calorimetry will be used to assess recognition patterns. Furthermore, structural analyses will be carried out via targeted enzymatic and chemical degradation, and electrophoretic assays. The interdisciplinary nature of the projects will attract a wide range of students interested in pursuing careers in the biomedical fields and will serve as a platform for a successful transition into their next academic/ professional step. The proposed activities will prepare undergraduate students to tackle challenges and solve important scientific problems while strengthening the PI?s research program, and department, at CU Denver.